Compositions and methods for enhancing sperm function

ABSTRACT

The disclosure provides, inter alia, methods of improving sperm function and related methods of fertilization, together with preparations of activated or potentiated sperm. The disclosure additionally provides articles of manufacture suitable for performing the methods provided by the invention. The methods provided by the disclosure, in some embodiments entail energy depletion with subsequent staged reintroduction of different energy sources.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/282,204, filed Feb. 21, 2019, which claims the benefit of U.S.Provisional Application No 62/773,471, filed Nov. 30, 2018, whichapplications are incorporated herein by reference in their entireties.

BACKGROUND

Male factor is a contributing factor for ˜50% of couples havingdifficulty conceiving. An important aspect of assisted reproduction isobtaining maximal function of male gametes (sperm) to help maximizefertilization. Accordingly, a need exists for media, compositions, andmethods for increasing sperm function, e.g., to facilitate assistedreproduction.

SUMMARY

In some embodiments, provided herein is a method of inducing increasedsperm function comprising; (a) incubating a mammalian sperm under energydepletion conditions for a time suitable to potentiate the mammaliansperm, (b) providing the potentiated mammalian sperm with an effectiveamount of a first energy source selected from: (i) a glycolytic energysource or (ii) a gluconeogenesis substrate, and (c) subsequentlyproviding the mammalian sperm from step (b) with an effective amount ofa second energy source, selected from: (i) the glycolytic energy sourceor (ii) the gluconeogenesis substrate, wherein the energy sourceprovided is not selected in step (b), thereby inducing increased spermfunction compared to a suitable control sperm.

In some embodiments, the suitable control sperm is an untreatedmammalian sperm, a potentiated mammalian sperm of step (a) provided withan effective amount of the glycolytic energy source or thegluconeogenesis substrate independently, or a potentiated mammaliansperm of step (a) provided with an effective amount of the glycolyticenergy source and the gluconeogenesis substrate simultaneously.

In some embodiments, the method is performed in vitro. In someembodiments of the method, step (c) is performed in vivo, in thereproductive tract of a female subject, by intrauterine insemination(IUI) of the mammalian sperm from step (b).

In some embodiments, the increased sperm function comprises an increasein motility as measured by computer assisted semen analysis (CASA). Insome embodiments, the increase in motility comprises an increase incurvilinear velocity of the mammalian sperm, increase in percentage ofhyperactivated sperm, increase in percentage of intermediate motilitysperm, or a combination thereof. In some embodiments, the increasedsperm function comprises an increase in sperm capacitation as measuredby a sperm-zona pellucida binding assay. In some embodiments, theincreased sperm function comprises an increase in ability of themammalian sperm to fertilize an egg as measured by a sperm penetrationassay. In some embodiments, the increased sperm function comprisesgeneration of an embryo with increased viability, and/or improvedimplantation relative to an embryo generated with a suitable controlsperm, or increased ability of an embryo to develop to at least a 2-celldevelopmental stage, blastocyst developmental stage or an offspringrelative to a embryo generated with a suitable control sperm.

In some embodiments, the mammalian sperm is a human, non-human primate,porcine, bovine, equine, ovine, canine, feline, or murine sperm. In someembodiments, the mammalian sperm is a human sperm. In some embodiments,the glycolytic energy source is glucose and the gluconeogenesissubstrate is pyruvate.

In some embodiments, provided herein is a preparation of spermcomprising the mammalian sperm with increased sperm function prepared bythe method described above.

In some embodiments, provided herein is a preparation of sperm preparedby: (a) incubating a mammalian sperm under energy depletion conditionsfor a time suitable to potentiate the mammalian sperm; and (b) providingthe potentiated sperm from step (a) with an effective amount of a firstenergy source and a second energy source in a serial manner, wherein thepreparation of sperm comprises an increase in one or more spermfunctions to a level greater than that obtained by providing thepotentiated sperm of step (a) with an effective amount of the firstenergy source or the second energy source independently, or providing aneffective amount of the first energy source and the second energy sourcesimultaneously.

In some embodiments, the one or more sperm function is selected fromcurvilinear velocity, amplitude of lateral head displacement, autophagy,sperm capacitation, percentage of hyperactivated sperm, percentage ofintermediate motility sperm and percentage of hyperactivated sperm andintermediate motility sperm or combinations thereof. In someembodiments, the first energy source is a glycolytic energy source andthe second energy source is a gluconeogenesis substrate, or the firstenergy source is the gluconeogenesis substrate and the second energysource is the glycolytic energy source. In some embodiments, themammalian sperm of step (a) is provided as a pool of two or moreejaculates.

In some embodiments, the mammalian sperm of step (a) is from asubfertile male or an oligospermic male. In some embodiments, themammalian sperm of step (a) is a human, non-human primate, porcine,bovine, equine, ovine, canine, feline, or murine sperm. In someembodiments, the mammalian sperm of step (a) is a human sperm.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety forall purposes, to the same extent as if each individual publication,patent, or patent application was specifically and individuallyindicated to be incorporated by reference. For example, all publicationsand patents mentioned herein are incorporated herein by reference intheir entirety for the purpose of describing and disclosing the kits,compositions, and methodologies that are described in the publications,which might be used in connection with the methods, kits, andcompositions described herein. The documents discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors described herein are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of the percentage of hyperactive and intermediatemotility sperm in control and starved (glucose, pyruvate, andlactate-free) conditions.

FIG. 2 is a bar graph of the percentage of hyperactive and intermediatemotility sperm in control and starved (glucose, pyruvate, andlactate-free) conditions following: addition of glucose and pyruvatetogether (Starve/Rescue simultaneous), glucose only (Starve/Glucoserescue), pyruvate only (Starve/Pyruvate only), 1 hour glucose+15 minutespyruvate (Starve/glucose rescue+15 minute pyruvate), or 1 hourpyruvate+15 minute Glucose.

FIG. 3 is an illustration of density gradient isolation of sperm coupledto certain exemplary embodiments of methods provided by the invention.

FIG. 4 is an illustration of swim-up isolation of sperm coupled tocertain exemplary embodiments of methods provided by the invention.

FIG. 5A is a bar graph of the percentage of intermediate motility sperm+/−SEM in 7 different donors N=20, *: p<0.05 relative to control asdetermined by t-test. Semen samples were obtained from healthyvolunteers.

FIG. 5B is a bar graph of the percentage of hyperactive motility sperm+/−SEM in 7 different donors N=20, *: p<0.05 relative to control asdetermined by t-test. Semen samples were obtained from healthyvolunteers.

FIG. 6A is a bar graph of the percentage of intermediate motility sperm+/−SEM. N=5, *: p<0.05 relative to control as determined by t-test.Semen samples were obtained from men seeking treatment for infertility.

FIG. 6B is a bar graph of the percentage of hyperactive motility sperm+/−SEM. N=5, *: p<0.05 relative to control as determined by t-test.Semen samples were obtained from men seeking treatment for infertility.

DETAILED DESCRIPTION OF THE INVENTION

Male factor is a contributing factor for ˜50% of couples havingdifficulty conceiving. Low sperm count is a recognized factor in maleinfertility. The World Health Organization defines low sperm count(oligospermia) as less than 15 million sperm per milliliter (Cooper etal., Human Reproduction Update, 16(3), 231-245, 2009). Other factorscontributing to male infertility or subfertility include low motility orabnormal morphology. An important aspect of assisted reproduction isobtaining maximal function of male gametes (sperm) to help maximizefertilization. Before fertilization, sperm must go through a series ofchanges to be able to fertilize the egg, a process called spermcapacitation. In vitro capacitation media, includes three components(albumin, calcium and bicarbonate) and initiate sperm capacitation.Sperm initially swim progressively with an almost symmetrical flagellarmovement. After different periods of time, which depend on the species,the straight sperm movement is replaced by an in-place helical movementknown as “hyperactivation”. While methods for activating sperm exist,they fail to achieve maximal sperm activation and therefore do notadequately address the impact of male factor in infertility.Accordingly, a need exists for media, compositions, and methods forincreasing sperm function, e.g., to facilitate assisted reproduction.

The present disclosure provides, inter alia, methods for increasingsperm function, preparations of sperm, methods of fertilization, andarticles of manufacture, e.g., useful for performing methods provided bythe disclosure. The invention is based, at least in part, on Applicant'ssurprising discovery that staged reintroduction of different energysources after a period of starvation achieves superior activation ofsperm.

Definitions

To facilitate an understanding of the present disclosure, a number ofterms and phrases are defined below.

The terms “increased”/“increase”, “increasing” or “enhance” or “promote”are all used herein to generally mean an increase; for the avoidance ofdoubt, the terms “increased”, “increase”, or “enhance”, mean an increaseof at least 5%, e.g., at least 10% as compared to a suitable control,for example an increase of at least about 10%, at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a suitable control, or at least about a2-fold, or at least about a 3 -fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to a suitablecontrol. The increase can be, for example, at least 10%, at least 20%,at least 30%, at least 40% or more, and is preferably to a levelaccepted as within the range of normal sperm from a mammalian malesubject without a given disease (e.g., male infertility, due to abnormalsperm function or oligospermia).

The terms, “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or“inhibit” are all used herein generally to mean a decrease. For example,“decrease”, “reduce”, “reduction”, or “inhibit” means a decrease by atleast 5%, e.g., 10% as compared to a suitable control, for example adecrease by at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% decrease (e.g., absent level or non-detectable level as comparedto a suitable control), or any decrease between 10-100% as compared to asuitable control. The decrease can be, for example, at least 10%, atleast 20%, at least 30%, at least 40% or more, than the range of normalfor an individual without a given disease.

As used herein, the term “effective amount” means the total amount ofthe active component(s) of a first energy source or a second energysource that is sufficient to cause a change on a detectable function ofthe mammalian sperm (e.g., sperm motility, curvilinear velocity,amplitude of lateral head displacement, autophagy, sperm capacitation,percentage of hyperactivated sperm, percentage of intermediate motilitysperm and percentage of hyperactivated sperm and intermediate motilitysperm, ability to fertilize an egg, and generation of an embryo). Whenapplied to an individual energy source, administered alone, the termrefers to that energy source alone. When applied to a combination, theterm refers to combined amounts of the first energy source and thesecond energy source that result in the effect, whether administered incombination, serially or simultaneously.

The term “an effective amount” includes within its meaning a sufficientamount of an energy source (e.g., a gluconeogenesis substrate orglycolytic energy source) to provide the desired effect. As it relatesto the present disclosure, the desired effect can be increase in one ormore sperm function or increase in fertilization. The exact amountrequired will vary depending on factors such as the mammalian spermspecies being treated, the age and general condition of the male subjectfrom whom the mammalian sperm is obtained, for example if the sperm isobtained from a sub-fertile mammalian subject. Thus, it is not possibleto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” may be determined by one of ordinaryskill in the art using only routine experimentation.

The term “spermatozoon” refers to a live reproductive cell from a malemammal. The term “spermatozoa” refers to a plurality of live malereproductive cells. Unless required otherwise by context, the plural andsingular forms are interchangeable. The term “sperm” is used as anabbreviation and refers to at least one spermatozoon.

As used herein, the term “ability to fertilize an egg” refers to abilityof a sperm (e.g., mammalian sperm) to penetrate an unfertilized egg(ovum) resulting in combination of their genetic material resulting inthe formation of a zygote. As it relates to the present disclosure, the“ability to fertilize” an egg can be ability to fertilize in vitroand/or in vivo. In some embodiments, ability to fertilize in vitrocomprises fertilization by Intracytoplasmic sperm injection (ICSI).

The term “embryo” is used herein to refer both to the zygote that isformed upon fertilization of an unfertilized egg by a mammalian sperm,to form a diploid totipotent cell, e.g. a fertilized egg and to theembryo that undergoes subsequent cell divisions to develop to 2-cellstage or greater (e.g., 4-cell stage, 16-cell stage, 32-cell stage, theblastocyst stage (with differentiated trophectoderm and inner cell mass)or development into an offspring).

As used herein, the term “ability to develop” refers to the ability orcapacity of an embryo to grow or develop. The terms may refer to theability or capacity of an embryo to reach at least the 2-celldevelopmental stage, the blastocyst developmental stage, implant intothe uterus, to develop to a full offspring, or be born live. The term“offspring” as used herein refers to a progeny of a parent, wherein theprogeny is an unborn fetus or a newborn.

The term “blastocyst” refers to an embryo, five or six days afterfertilization, having an inner cell mass, an outer cell layer called thetrophectoderm, and a fluid-filled blastocele cavity containing the innercell mass from which the whole of the embryo is derived. Thetrophectoderm is the precursor to the placenta. The blastocyst issurrounded by the zona pellucida which is subsequently shed when theblastocyst “hatches.” The zona pellucida, composed of a glycoproteincoat, surrounds the oocyte from the one-cell stage to the blastocyststage of development. Prior to embryo attachment and implantation, thezona pellucida is shed from the embryo by a number of mechanismsincluding proteolytic degradation. The zona pellucida functionsinitially to prevent entry into the oocyte by more than one sperm, thenlater to prevent premature adhesion of the embryo before its arrivalinto the uterus.

As used herein, the term “enriched” refers to a composition or fractionor preparation wherein an object species has been partially purifiedsuch that the concentration of the object species is substantiallyhigher than the naturally occurring level of the species in a finishedproduct or preparation without enrichment.

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

i. X is at least 100;ii. X is at least 200;iii. X is at least about 100; andiv. X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

i. X being administered on between day 1 and day 2;ii. X being administered on between day 2 and day 3;iii. X being administered on between about day 1 and day 2;iv. X being administered on between about day 2 and day 3;v. X being administered on between day 1 and about day 2;vi. X being administered on between day 2 and about day 3;vii. X being administered on between about day 1 and about day 2; andviii. X being administered on between about day 2 and about day 3.

All these different combinations are contemplated by the rangesdisclosed throughout. All disclosed ranges should be interpreted in thismanner, whether it refers to an administration of a therapeutic agent orreferring to days, months, years, weight, dosage amounts, etc., unlessotherwise specifically indicated to the contrary.

Sperm Function

In some embodiments, provided herein is a method for increasing spermfunction. The method comprises incubating a mammalian sperm under energydepletion for a time suitable to potentiate the mammalian sperm,providing the potentiated mammalian sperm with an effective amount of afirst energy source selected from: (i) a glycolytic energy source or(ii) a gluconeogenesis substrate, and subsequently providing themammalian sperm from step (b) with an effective amount of a secondenergy source, selected from: (i) the glycolytic energy source or (ii)the gluconeogenesis substrate, wherein the energy source provided is notthe one selected as first energy source, thereby inducing increasedsperm function compared to a suitable control sperm. In someembodiments, the method is performed in vitro. In some embodiments, theproviding of the second energy source is performed in vivo, for example,by cervival or intrauterine insemination of the sperm which has beenpreviously incubated under energy depletion and provided a first energysource. Increased sperm function includes one or more of: increasedmotility such as the percentage of sperm in a population exhibitinghyperactivation and/or intermediate motility as assessed by CASAnova(see Goodson et al., 2017, Biol. Reprod. 97:698-708;doi:10.1093/biolre/iox120), increased autophagy, increased capacitation,and increased rates of fertilization, e.g., development to at least twocells, blastocyst development, or live birth. Accordingly, in someembodiments, sperm function can be sperm motility, curvilinear velocity,amplitude of lateral head displacement, autophagy, sperm capacitation,percentage of hyperactivated sperm, percentage of intermediate motilitysperm and percentage of hyperactivated sperm and intermediate motilitysperm, ability to fertilize an egg, generation of an embryo. In someembodiments, the embryo generated by the sperm with increased functioncomprises one or more characteristics selected from increased viability,increased implantation, increased ability to develop to a at least a2-cell developmental stage, blastocyst developmental stage or anoffspring.

In some embodiments, the first and second energy sources are provided ina serial manner (e.g., providing a first energy source and subsequentlyproviding a second energy source). In some embodiments, the first andsecond energy sources are provided simultaneously. An increase in one ormore sperm functions, as contemplated herein, constitutes an increase inthe one or more sperm functions relative to a suitable control sperm. Insome embodiments, the one or more sperm functions can be increased by atleast or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%,or 100%, 200%, 300% or more. In some embodiments, the one or more spermfunctions can be increased by from 10% to 200%, from 25% to 150%, from50% to 100%, or from 70% to 90%.

Provided herein, are methods to increase sperm function and preparationof sperm comprising increased function relative to a suitable controlsperm. As it relates to the present disclosure, sperm “activity” and/or“function” encompass physiological processes such as, for example, spermmotility, sperm tropism (namely, the tendency of sperm to move towardsor away from certain stimuli), and ability to fertilize an egg. Theterms “activity” and/or “function” can further include processes whichoccur prior to, during fertilization and/or interaction with the egg (ormembranes/layers thereof)—such processes may include, for example spermcapacitation and acrosomal activity, and/or processes afterfertilization of egg, for example, formation of an embryo. In someembodiments, the embryo exhibits increased (longer) viability, improvedimplantation, and/or ability to develop to a 2-cell stage, a blastocyst,or to an offspring resulting in live birth.

Exemplary methods to measure an increase in sperm function may beassessed by motility, mucus penetration, oocyte fertilization orsubsequent embryonic development and the like. Methods to determinesperm function are well known in the art, see for example, SS. VasanIndian J Urol. 2011 January-March; 27(1): 41-48.

Sperm Motility

With regard to sperm motility, one of skill will appreciate that theterm “motility” not only relates to general movement, but may be appliedto other aspects of motility such as, for example, the speed of movementof a sperm cell and/or any increase or decrease in the proportion ofmoving sperm cells in any given population. As such, the methodsdisclosed herein may be used not only to increase sperm motility, butalso to increase the speed of movement of a sperm cell and/or theproportion (percentage) of moving cells in any given population ofsperm.

Motility of sperm is expressed as the total percent of motile sperm, orthe velocity of sperm that are motile. These measurements may be made bya variety of assays, but are conveniently assayed in one of two ways.Either a subjective visual determination is made using a phase contrastmicroscope when the sperm are placed in a hemocytometer or on amicroscope slide, or a computer assisted semen analyzer is used. Underphase contrast microscopy, motile and total sperm counts are made andspeed is assessed as fast, medium or slow. A second method of assessingsperm motility is by using a computer assisted semen analyzer (HamiltonThom, Beverly, Mass.), the motility characteristics of individual spermcells in a sample are objectively determined. Briefly, a sperm sample isplaced onto a slide or chamber designed for the analyzer. The analyzertracks individual sperm cells and determines motility and velocity ofthe sperm. Data is expressed as percent motile, and measurements areobtained for path velocity and track speed as well.

Accordingly, the term “motility” encompasses percentage of motile spermwhich can be the percentage of the total number of sperm assessed thatfall within all World Health Organization (WHO) categories of motilityexcept the category designated “no motility” regardless of velocity ordirectionality as discussed in Cooper et al. Human Reproduction update,Vol 16, No 3 pp 231-245, 2010. Manual counting classifies sperm cellsinto 4 categories (immotile, locally motile, non linear and linearmotile) using qualitative subjective criteria of selection.

The term “motility” encompases percentage of motile sperm i.e., thepercentage of total number of sperm assessed in a population exhibitingprogressive motility, hyperactivated motility and/or intermediatemotility based on Computer assisted sperm analysis (For example, asassessed by CASAnova; see Goodson et al, 2017, Biol. Reprod.97:698-708).

The methods disclosed herein can increase percentage of progressivemotility sperm, i.e., percentage of sperm exhibiting linear movementfrom one point to another, with turns of the head of less than 90degrees from sperm that are otherwise non-progressive, i.e., sperm thatmove but do not make forward progression. In some embodiments, themethods disclosed herein can increase percentage of intermediatemotility sperm. Intermediate motility sperm is characterized by movementthat is similar to progressive vigorous motility, but has a largervariance from the path and turns of the sperm head of approximately 90degrees, such as an oscillating movement. In some embodiments, theincreased motility comprises increase in percentage of activatedhyperactive sperm, also known as hyperactivated sperm. Hyperactivatedsperm motility is characterized by sperm that have a high amplitude,asymmetrical beating pattern of the flagellum. Hyperactivated motilityis characterized by vigorous movement with many seemingly randomvariations without a well-defined progressive path and turns of thesperm head of greater than 90 degrees. Hyperactivated sperm motility ismore vigorous and short term than progressive motility. Biologically,hyperactivated sperm motility is important to enable sperm to traversethe egg outer investments prior to fertilizing the mature egg. In someembodiments, the methods disclosed herein can increase percentage ofhyperactivated sperm and intermediate motility sperm in a givenpopulation of sperm.

It should be understood that other standardized measures of spermmotility parameters can also be used. Other measures of sperm motilityinclude “velocity” and “linearity” which can be assessed using automaticsemen analyzers. In some embodiments, the methods disclosed herein canincrease sperm function comprising increase in average path velocity(VAP), straight-line velocity (VSL), curvilinear velocity (VCL),amplitude of lateral head displacement (ALH) and beat cross frequency(BCF) or other movement parameters of the sperm including parametersknown to those of skill in the art. Curvilinear velocity (VCL) is themeasure of the rate of travel of the centroid of the sperm head over agiven time period. Average path velocity (VAP) is the velocity along theaverage path of the spermatozoon. Straight-line velocity (VSL) is thelinear or progressive velocity of the cell. Linearity of forwardprogression (LIN) is the ratio of VSL to VCL and is expressed aspercentage. Amplitude of lateral head displacement (ALH) of the spermhead is calculated from the amplitude of its lateral deviation about thecell's axis of progression or average path. Methods of measuring spermmotility by CASA are well known in the art, see for example,WO2012061578A2, An increase in sperm motility, as contemplated herein,constitutes an increase in the motility of sperm relative to a suitablecontrol sperm.

In some embodiments sperm with increased motility are provided that arethe product of a process comprising incubating sperm in energy depletionconditions to potentiate the sperm, followed by providing thepotentiated sperm with a first energy source and a second energy source.In some embodiments, the first and second energy sources are providedsimultaneously. In some embodiments, the first and second energy sourcesare provided serially. In some embodiments, the increase in spermmotility can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 99% relative to a suitable control sperm. In some embodiments,the increase in sperm motility can be at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%. In someembodiments, the increase in sperm motility can be by a factor of atleast 10, at least 100, at least 1,000, at least 10,000. In someembodiments, the sperm motility can be increased by from 10% to 200%,from 25% to 150%, from 50% to 100%, or from 70% to 90%. In someembodiments, the increased sperm function or increased sperm motilitycan be an increase in percentage of hyperactivated sperm. In someembodiments, the increased sperm function or increase in sperm motilitycan be an increase in percentage of intermediate motility sperm. In someembodiments, the increased sperm function or increased sperm motilitycan be an increase in percentage of progressive motility sperm. In someembodiments, the increased sperm function or increased sperm motilitycan be an increase in percentage of the hyperactivated sperm andintermediate motility sperm. In some embodiments, the level ofhyperactivated sperm, progressive motililty sperm, intermediate motilitysperm or a combination thereof is increased so that hyperactivatedsperm, progressive motililty sperm, intermediate motility sperm or acombination thereof comprise at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%,7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%,13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%,18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or moreof the total sperm in a preparation. An increase in sperm motility isindicative of increased sperm function.

Sperm Capacitation

In some embodiments the increased sperm function comprises an increasein sperm capacitation. “Sperm capacitation” refers to the sperm havingthe ability to undergo acrosomal exocytosis and binding to andpenetrating through the zona pellucida of an unfertilized egg.Completion of capacitation is manifested by the ability of sperm to bindto the zona pellucida and to undergo ligand-induced acrosomal reaction.Methods to determine sperm capacitation are known in the art, forexample, the most common sperm-zona pellucida binding tests currentlyutilized are the hemizona assay (or HZA) and a competitive intact-zonabinding assay. A hemizona assay measures the ability of sperm to undergocapacitation and bind to an oocyte. Sperm is incubated with dead oocyteswhich are surrounded by the zona pellucida, an acellular coating ofoocytes. Capacitated sperm bind to the zona and the number of spermbinding is counted microscopically. This number correlates with thenumber of normal capacitated sperm in a sample and with fertility of asperm sample. For example, see Cross NL et al. Gamete Res. 1986;15:213-26.

In some embodiments, sperm with increased capacitation are provided thatare the product of a process comprising incubating sperm in energydepletion conditions to potentiate the sperm, followed by providing thepotentiated sperm with a first energy source and a second energy sourcesimultaneously, or serially. In some embodiments, the increase in spermcapacitation can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 99% relative to a suitable control sperm. In someembodiments, the increase in sperm capacitation can be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Insome embodiments, the increase in sperm capacitation can be by a factorof at least 10, at least 100, at least 1,000, at least 10,000. In someembodiments, the sperm capacitation can be increased by from 10% to200%, from 25% to 150%, from 50% to 100%, or from 70% to 90%. In someembodiments, the level of sperm capacitation is increased so thatcapacitated sperm can comprise at least about 5%, 5.5%, 6.0%, 6.5%,7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%,12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%,17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50%or more of the total sperm in a preparation. An increase in spermcapacitation is indicative of increased sperm function.

Fertilizing Ability

In some embodiments, the sperm function comprises ability of the spermto fertilize an egg. The fertilizing ability of a sperm can bedetermined, for example, by a sperm penetration assay. The spermatozoapenetration assay (SPA) utilizes the golden hamster egg, which isunusual in that removal of its zona pellucida results in loss of allspecies specificity to egg penetration. This test is conducted todetermine the ability of sperm to penetrate into the oocyte (Rogers etal., Fert. Ster. 32:664, 1979). Briefly, commercially available zonafree hamster oocytes can be used (Fertility Technologies, Natick,Mass.). Hamster oocytes are suitable in this assay for sperm of anyspecies. Sperm are incubated for 3 hours with the hamster oocytes.Following incubation, oocytes are stained with acetolacmoid orequivalent stain and the number of sperm penetrating each oocyte iscounted microscopically. Another parameter of sperm fertilizing abilityis the ability to penetrate cervical mucus. This penetration test can bedone either in vitro or in vivo. Briefly, in vitro, a commercial kitcontaining cervical mucus (Tru-Trax, Fertility Technologies, Natick,Mass.), typically bovine cervical mucus, is prepared. Sperm are placedat one end of the track and the distance that sperm have penetrated intothe mucus after a given time period is determined. Alternatively, spermpenetration of mucus may be measured in vivo in women. In an embodimentsperm with increased fertilizing ability are provided that are theproduct of a process comprising incubating sperm in energy depletionconditions to potentiate the sperm, followed by providing thepotentiated sperm with a first energy source and a second energy sourcesimultaneously, or serially. In some embodiments, the increase infertilizing ability can be more than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 99% relative to a suitable control sperm. In someembodiments, the increase in fertilizing ability can be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Insome embodiments, the increase in fertilizing ability can be by a factorof at least 10, at least 100, at least 1,000, at least 10,000. In someembodiments, the fertilizing ability can be increased by from 10% to200%, from 25% to 150%, from 50% to 100%, or from 70% to 90%. In someembodiments, the level of fertilizing ability is increased so that thenumber of sperm able to fertilize an egg is at least about 5%, 5.5%,6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%,11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%,16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%,35%, 40%, 50% or more of the total sperm in a preparation. An increasein fertilizing ability is indicative of increased sperm function andincreased fertilization.

Generating Embryos

In some embodiments, sperm function comprises generating an embryo. Insome embodiments, the sperm with increased function prepared by methodsherein is provided access to an egg to promote fertilization, whereinpromoting fertilization can comprise generation of an embryo. In someembodiments, the sperm with increased function prepared by the methodsherein is provided access to an egg in vitro, thereby generating theembryo in vitro. In some embodiments, the sperm with increased functionprepared by the methods disclosed herein is provided access to an egg invivo by IUI of the sperm, thereby generating the embryo in vivo. In someembodiments, the sperm which has been incubated under energy deletionconditions and provided with first energy source is inseminated in thereproductive tract of a female subject such that providing the secondenergy source and providing access to an egg to generate an embryooccurs in vivo. In some embodiments, where the embryo is generated invitro, the embryo can be cryopreserved for later use or can be furthercultured in vitro to enable embryonic development. In some embodiments,the embryo is developed to at least a two cell stage prior tocryopreserving and/or implantation into a female subject. In someembodiments, the embryo is developed to a developmental stage greaterthan the two-cell stage in vitro prior to further processing. In someembodiments, the embryo is developed to a blastocyst stage in vitroprior to further processing (e.g., cryopreservation or implantation intoa female subject to develop into a full offspring). For in vitroincubation and culture of embryos during via assisted reproductivetechnologies (ART) procedures, a range of suitable media are available,the types and compositions of which are well known to those of skill inthe art. Preferably the culture medium contains at least water, salts,nutrients, essential amino acids, vitamins and hormones, and may alsoinclude one or more growth factors. A variety of suitable culture mediais commercially available, for example Earle's media, Ham's F10 mediaand human tubal fluid (HTF) media. The present disclosure alsocontemplates the co-culture in vitro of embryos on a layer of ‘feedercells’ by methods known in the art. Appropriate ‘feeder cells’ forco-culture may include, for example, bovine oviductal cells or humantubal epithelial cells.

Those of skill in the art will appreciate that the advantages offered bythe sperm with increased function prepared by the methods disclosedherein are not limited to increasing fertilization. Rather the methodsand preparation of the present invention are equally applicable astreatment to promote fertilization, whether the embryos are produced invitro via assisted reproductive technologies (ART) or in thereproductive tract of the animal. The methods of the present inventionare applicable to improving fertilization, embryo viability, embryoimplantation and pregnancy rates in assisted or otherwise unassistedpregnancies. Embodiments of the present disclosure also provide formethods of increasing the fertilizing ability of sperm in male animals.

In the context of this specification, the terms “embryo with increasedviability” and “embryo with longer viability” mean an increase orenhancement in the likelihood of survival of an embryo(s) which has beengenerated by the mammalian sperm of the methods and preparationdisclosed herein, for example, a mammalian sperm with one or moreincreased sperm function, compared to the likelihood of survival of anembryo(s) which has been generated by a suitable control sperm. In someembodiments, the embryo is generated by an assisted reproductivetechnology e.g., IVF or ICSI. In some embodiments, the embryo isgenerated in vivo in the reproductive tract of a female mammaliansubject by artificial insemination.

For the purposes of the present disclosure, embryo viability may bereflected in a number of indicators. For example, increased embryoviability may result in increased embryo implantation rates followingfertilization, decreased pre- and post-implantation embryo lethality,increased clinical pregnancy rates or increased birth rates. The presentdisclosure therefore also relates to methods of preventing apoptosis orretarded development in embryos and to methods of increasing pregnancyrates in animals. The embryo viability can refer to viability of anembryo in vitro or in vivo.

In some embodiments, sperm with ability to generate an embryo withincreased viability is provided that are the product of a processcomprising incubating sperm in an energy depletion conditions topotentiate the sperm, followed by providing the potentiated sperm with afirst energy source and a second energy source simultaneously, orserially. In some embodiments, providing the sperm with increasedfunction access to an egg promotes fertilization. In some embodiments,promoting fertilization comprises generation of an embryo(s) withincreased viability. In some embodiments, the increase in viability ofembryo generated by the sperm prepared by methods herein upon access toan egg can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 99% relative to an embryo generated by a suitable control sperm.In some embodiments, the increase in embryo viability can be at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least95%. In some embodiments, the increase in embryo viability can be by afactor of at least 10, at least 100, at least 1,000, at least 10,000. Insome embodiments, the embryo viability can be increased by from 10% to200%, from 25% to 150%, from 50% to 100%, or from 70% to 90%. In someembodiments, the level of sperms that can generate an embryo withincreased viability is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%,8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%,13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%,18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of thetotal sperm in a preparation. Generation of an embryo with increasedviability is indicative of increased sperm function and/or increasedfertilization.

Typically the cleavage stage of embryo occurs during the first threedays of culture. The in vitro generated embryo is transferred to afemale subject by embryo transfer. “Embryo transfer” is the procedure inwhich one or more embryos and/or blastocysts are placed into the uterusor fallopian tubes. In the traditional IVF process, embryos aretransferred to the uterine cavity two days after fertilization when eachembryo is at the four (4) cell stage or three days after fertilizationwhen the embryo is at the eight (8) cell stage. It has been recognizedthat it may be desirable to use embryos at the blastocyst stage whenreached at day five to seven of culture. The present disclosure allowsfor embryo transfer at any time along the spectrum of embryo/blastocystdevelopment. Through visual observation, such as by with the use ofmicroscopy, blastocysts or embryos are considered ready to betransferred to the uterus when the blastoceol cavity is clearly evidentand comprises greater than 50% of the volume of the embryo. In an invivo environment, this stage would normally be achieved four to fivedays after fertilization, soon after the embryo has traversed thefallopian tube and arrives in the uterus. Embryonic developmental stagecan be determined by visual observation of the embryo using microscopy(for example, Nikon Eclipse TE 2000-S microscope), the embryo willdisplay certain determined physical or morphological featuressimultaneously before it is implanted into the uterus. The state ofblastocyst maturity will be determined to be the range II AB-VI AAaccording to classification of Gardner et al, 1998.

The methods disclosed herein result in generation of embryos withincreased rate of progressing to 2-cell developmental stage, blastocystdevelopmental stage, or development to an offspring and live birth. Insome embodiments, sperm which can generate an embryo with ability todevelop through normal developmental stages (e.g., 2 cell stage,blastocyst stage, development into an offspring and live birth) isprovided that are the product of a process comprising incubating spermin an energy depletion conditions to potentiate the sperm, followed byproviding the potentiated sperm with a first energy source and a secondenergy source simultaneously, or serially. In some embodiments,providing the sperm with increased function access to an egg promotesfertilization. In some embodiments, promoting fertilization comprisesgeneration of embryos with increased ability to develop through normaldevelopmental stages (e.g., 2 cell stage, blastocyst stage, developmentinto an offspring and live birth). In some embodiments, increase in rateof an embryo progressing through normal developmental stages, generatedby the sperm prepared by methods can be more than about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 99% relative to an embryo generated bysuitable control sperm. In some embodiments, the increase in rate of anembryo progressing through normal developmental stages can be at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least95%. In some embodiments, the increase in rate of an embryo progressingthrough normal developmental stages can be by a factor of at least 10,at least 100, at least 1,000, at least 10,000. In some embodiments, therate of embryo progressing through normal developmental stages can beincreased by from 10% to 200%, from 25% to 150%, from 50% to 100%, orfrom 70% to 90%. In some embodiments, the level of sperms that cangenerate an embryo with ability to progress through normal developmentalstages is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%,14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%,19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% or more of the totalsperm in a preparation. Generation of an embryo with ability to progressthrough one or more normal developmental stages is indicative ofincreased sperm function and/or increased fertilization.

In vivo, an embryo attaches or implants to a wall of the uterus, createsa placenta, and develops into a fetal offspring during gestation untilchildbirth. Testing to determine whether one or more embryos haveimplanted into the endometrium, i.e, whether the procedure has resultedin successful pregnancy inception, is performed two weeks after transferusing blood tests on b-hCG (human chorionic gonadotropin), for example,and other techniques commonly known in the art. U.S. Pat. No. 4,315,908to Zer et al. sets forth a method for detecting hCG in the urine byradioimmunoassay. U.S. Pat. No. 8,163,508 to O'Connor et al. provides amethod and a kit for predicting pregnancy in a subject by hCG method bydetermining the amount of an early pregnancy associated isoform of hCGin a sample. Such methods of diagnosis and others are useful within thescope of the disclosure.

In some embodiments, sperm with ability to generate an embryo withimproved implantation rate or improved rate of pregnancy is providedthat are the product of a process comprising incubating sperm in anenergy depletion conditions to potentiate the sperm, followed byproviding the potentiated sperm with a first energy source and a secondenergy source simultaneously, or serially. In some embodiments,providing the sperm with increased function access to an egg promotesfertilization. In some embodiments, promoting fertilization comprisesgeneration of an embryo with improved implantation rate or improved rateof pregnancy. In some embodiments, the increase in implantation rate ofan embryo generated by the sperm prepared by methods herein or pregnancyrate upon implantation of an embryo can be more than about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% relative to an embryogenerated by a suitable control sperm. In some embodiments, the increasein an embryo implantation rate or pregnancy rate can be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Insome embodiments, the increase in rate of embryo implantation or rate ofpregnancy can be by a factor of at least 10, at least 100, at least1,000, at least 10,000. In some embodiments, the embryo implantation orpregnancy rate can be increased by from 10% to 200%, from 25% to 150%,from 50% to 100%, or from 70% to 90%. In some embodiments, the level ofsperms that can generate an embryo with increased implantation rate orimproved pregnancy rate is at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%,7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%,13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%,18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%, 40%, 50% more ofthe total sperm in a preparation. Generation of embryos with improvedimplantation (i.e., increased rate of implantation) or increasedpregnancy rate upon implantation is indicative of increased spermfunction and/or increased fertilization.

Autophagy

In some embodiments, the increased sperm function comprises an increasein autophagy. Methods to determine an increase in autophagy are known inthe art. For example, an increase in autophagy can be determined byincrease in one or more of autophagy marker proteins. The detection ofincrease in marker protein can be done by conventional methods such asimmunoblotting. Non-limiting examples of autophagy marker proteinsinclude, Atg 5, Atg 16, p62, LC3-II, AMPK, m-TOR and Beclin 1. LC3-IIhas been widely used to study autophagy and it has been considered as anautophagosomal marker in mammals. A ratio of LC3-II/LC3-I can be used asa determinant of increase in autophagy. An increase in levels of one ormore autophagy marker proteins (e.g., Atg 5, Atg 16, p62 and LC3-II,AMPK, m-TOR and Beclin 1), and/or an increase in ratio of LC3-II/LC3-Ican be indicative of increase in sperm function.

In some embodiments, sperm with increased autophagy are provided thatare the product of a process comprising incubating sperm in energydepletion conditions to potentiate the sperm, followed by providing thepotentiated sperm with a first energy source and a second energy sourcesimultaneously, or serially. In some embodiments, the increase inautophagy can be more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 99% relative to a suitable control sperm. In some embodiments,the increase in sperm autophagy can be at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%. In someembodiments, the increase in sperm autophagy can be by a factor of atleast 10, at least 100, at least 1,000, at least 10,000. In someembodiments, the sperm autophagy can be increased by from 10% to 200%,from 25% to 150%, from 50% to 100%, or from 70% to 90%. In someembodiments, the level of sperm autophagy is increased so that spermwith increased autophagy can comprise at least about 5%, 5.5%, 6.0%,6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%,12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%,17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20%, 25%, 30%, 35%,40%, 50% or more of the total sperm in a preparation. An increase insperm autophagy is indicative of increased sperm function.

Starvation

“Energy depletion” means suppressing or restricting the energetic outputof a cell whether by depletion, reduction (below an effective amount),or removal of such energy sources or inhibition of enzymatic or importmachinery. In some embodiments one or more of glycolysis,gluconeogenesis, Kreb's cycle, or oxidative phosphorylation areinhibited in the energy depletion and, in particular embodiments, theenergy depletion includes glycolytic energy depletion. Exemplaryconditions of glycolytic energy depletion include removing substantiallyall of glycolytically-liable sugar, such as glucose (other embodimentscan include, mannose, fructose, dextrose, sucrose, and combinationsthereof, including combinations with glucose), in the sperm's medium orreducing the concentration of glycolytically-liable sugar, or usinginhibitors of glycolysis, gluconeogenesis, or importers ofglycolytically-liable sugars. As glucose is a primary energy source ofsperm, in preferred embodiments, the energy depletion is glucose energydepletion (including starvation), which further entails depletion(including starvation) of gluconeogenesis substrates (including, e.g.,pyruvate), and Kreb's cycle substrates (acetyl CoA, citrate, isocitrate,alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, andoxaloacetate).

In some embodiments, the energy depletion comprises a low glucoseconcentration, e.g., less than about: 0.5, 0.4, 0.3, 0.2, 0.1, 0.09,0.08, 0.07, 0.06, 0.05, 0.04, 0.03, mM glucose, or less, such as lessthan about: 0.02 or 0.01 mM, e.g., less than about 0.01 mM. In someembodiments the energy depletion means a substantially glucose-freecondition. The invention provides methods entailing staged provision ofeffective amounts of first and second energy sources and the skilledartisan will appreciate that in some embodiments encompassed within theinvention, sub-effective amounts of a glycolytic energy source are anenergy depletion and, for example, the foregoing low glucoseconcentrations can be employed in such embodiments as an energydepletion.

In some embodiments, the energy depletion comprises a low pyruvateconcentration, e.g., less than about 0.15, 0.10, 0.09, 0.08, 0.07, 0.06,0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.003, 0.002 mM, or less. In someembodiments the energy depletion means a substantially pyruvate-freecondition. As noted above and exemplified with glucose for a glycolyticenergy source, the skilled artisan will also appreciate that in someembodiments encompassed within the invention sub-effective amounts of agluconeogenesis substrate are an energy depletion and, for example, theforegoing low pyruvate concentrations can be employed in suchembodiments as an energy depletion

In some particular embodiments, the energy depletion comprises acondition substantially free of carbon sources, such as low glucoseconcentration and low pyruvate concentration, e.g., a substantiallyglucose-free and substantially pyruvate-free condition.

In some embodiments, the energy depletion is for at least about: 10, 20,30, 40, 50, 60 minutes, e.g., at least about: 30, 40, 45, 50, 55, 60,90, 120, 150, or 180 minutes or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 hours.

Energy depletion consonant with the invention potentiates the sperm.“Potentiate” or “potentiating” sperm means to condition sperm such that,upon a suitable induction, e.g., removing or reversing the energydepletion and, e.g., incubating the sperm in capacitation conditions orstaged energy reintroduction, the sperm rapidly recover motility, suchas one or more of: an increased proportion of hyperactivated,intermediate, or progressive motility sperm (or an increased proportionof a combination of two (such as hyperactivated and intermediate) or allthree), and/or increased curvilinear velocities.

Staged Energy Reintroduction

Following energy depletion sufficient to potentiate the sperm, aneffective amount of a first and then an effective amount of a secondenergy source is provided to the potentiated sperm. In some embodiments,the time between providing an effective amount of a first energy sourceafter potentiating the sperm and providing an effective amount of asecond energy source is at least about: 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 minutes, e.g., at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, e.g., at leastbetween about: 5-15 minutes. In some embodiments, the time betweenproviding an effective amount of a first energy source afterpotentiating the sperm and providing an effective amount of a secondenergy source is longer, such as at least 2, 3, 4, or 5 hours, or more.

In some embodiments, the gluconeogenesis substrate is pyruvate, e.g., ata concentration of about: 0.15-0.66 mM, e.g., about 0.20-0.50 mM, suchas about 0.25-0.40 mM, or about 0.30 mM. The forgoing concentrations areexemplary effective amounts of a gluconeogenesis substrate, for example,when provided as either a first or second energy source in the methodsprovided by the invention. The skilled artisan will recognize othereffective amounts of gluconeogenesis substrates by virtue of theirability to increase sperm function consonant with the teachings of theinvention. In some embodiments, the first energy source is agluconeogenesis substrate, such as pyruvate. In some embodiments, thesecond energy source is a gluconeogenesis substrate, such as pyruvate.

In some embodiments, the glycolytic energy source is glucose, e.g., at aconcentration of about: 0.6 mM -10.0 mM, 1.0-7.0 mM, 2.5-7.0 mM, 3.5-6.5mM or 5 mM, e.g., at least about 1, 2, 3, or 4 mM. The forgoingconcentrations are exemplary effective amounts of a glycolytic energysource, for example, when provided as either a first or second energysource in the methods provided by the invention. The skilled artisanwill recognize other effective amounts of glycolytic energy sources byvirtue of their ability to increase sperm function consonant with theteachings of the invention. In some embodiments, the first energy sourceis a glycolytic energy source, such as glucose. In some embodiments thesecond energy source is a glycolytic energy source, such as glucose. Insome embodiments, the first energy source is a glycolytic energy source,such as glucose, while the second energy source is a gluconeogenesissubstrate, such as pyruvate.

An additional condition regulated in some embodiments of the methodsprovided by the invention is the osmolarity (mOsm) or osmolality(mOsm/kg). In some embodiments, the method is performed at an osmolarity(or osmolality) ranging from between about: 200 -280 mOsm (mOsm/kg),e.g., between about: 220-260, 225-255, 230-250 mOsm (mOsm/kg) duringenergy depletion, optionally, wherein upon addition of the first orsecond energy source, the osmolarity (or osmolality) is increased to atleast about: 270, 275, 280, 285, 290, or 295 mOsm (mOsm/kg).

In some embodiments of the methods provided by the invention, additionalcomponents are provided to the sperm. For example, other componentsupstream and downstream of glycolysis such as NADH , NAD⁺, citrate, AMP,ADP, or a combination thereof are added in combination with at least thefirst energy source or the second energy source.

Some embodiments of the methods provided by the invention includeassessment of the sperm. For example, in some embodiments, the methodsinclude one or more quantitative assessments of sperm motility, e.g., byCASA, and/or measuring sperm quality, such as DNA fragmentation (e.g.,by TUNEL), lipid peroxidation, reactive oxygen species, or a combinationthereof

The methods provided by the invention achieve increased sperm function.In some embodiments, relative to a suitable control sperm. In someembodiments the suitable control is sperm in standard capacitationmedium (C-HTF), without a starvation step, while in some embodiments,the suitable control is sperm in standard capacitation medium (C-HTF)following a three hour starvation—e.g., starvation and reintroduction ofeffective amounts of energy sources without staging reintroduction ofthe energy sources.

Mammalian Sperm

The methods disclosed herein comprise increasing one or more functionsof a sperm. The present disclosure also relates to promotingfertilization. Preparations of sperm with increased function are alsoprovided. As used herein, the sperm can be from a vertebrate, preferablya mammal. Accordingly, the sperm of the present disclosure can be amammalian sperm.

Mammals include, without limitation, humans, primates, rodents, wild ordomesticated animals, including feral animals, farm animals, sportanimals, and pets. Rodents include, for example, mice, rats, woodchucks,ferrets, rabbits and hamsters. Domestic and game animals include, forexample, cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, and canine species, e.g., dog, fox, wolf, avian species,e.g.,, chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.The mammalian sperm can be from a non-human mammal including, anungulate, such as an even-toed ungulate (e.g., pigs, peccaries,hippopotamuses, camels, llamas, chevrotains (mouse deer), deer,giraffes, pronghorn, antelopes, goat-antelopes (which include sheep,goats and others), or cattle) or an odd-toed ungulate (e.g., horse,tapirs, and rhinoceroses), a non-human primate (e.g., a monkey,chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g.,Rhesus.), a Canidae (e.g., a dog) or a cat. The mammalian sperm can befrom a member of the Laurasiatheria superorder. The Laurasiatheriasuperorder can include a group of mammals as described in Waddell etal., Towards Resolving the Interordinal Relationships of PlacentalMammals. Systematic Biology 48 (1): 1-5 (1999). The Members of theLaurasiatheria superorder can include Eulipotyphla (hedgehogs, shrews,and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora(carnivores), Cetartiodactyla (artiodactyls and cetaceans), Chiroptera(bats), and Pholidota (pangolins). A member of Laurasiatheria superordercan be an ungulate, e.g., an odd-toed ungulate or even-toed ungulate. Anungulate can be a pig. The mammalian sperm can be from a member ofCarnivora, such as a cat, or a dog. In some embodiments, the mammaliansperm is a human, non-human primate, porcine, bovine, equine, ovine,canine, feline, or murine sperm. In some embodiments, the mammaliansperm is a human sperm.

In some embodiments, the mammalian sperm is from a healthy male mammal.In some embodiments, the mammalian sperm is from a male suffering fromsperm dysfunction, for example, low sperm count, reduced motility ofsperm, and abnormal morphology of sperm. In some embodiments, themammalian sperm can be from a subfertile male or an oligospermic male.The mammalian sperm can be from a male suffering from, for example,oligospermia, Teratozoospermia, Asthenozoospermia, orOligoasthenoteratozoospermia. Oligospermia refers to a conditioncharacterized by sperm concentration of <20 million/ml.Asthenozoospermia refers to a condition characterized by reduced spermmotility. Teratozoospermia refers to a condition characterized bypresence of sperm with abnormal morphology. Oligoasthenoteratozoospermiarefers to a condition that includes oligozoospermia (low number ofsperm), asthenozoospermia (poor sperm movement), and teratozoospermia(abnormal sperm shape). In some embodiments, the sperm is obtained froma subfertile male or an oligospermic male, e.g., having a sperm countbelow about: 20, 19, 18, 18, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 million sperm per milliliter, e.g., less than 15 million spermper milliliter.

In some embodiments, the sperm are enriched (or isolated) from semenprior to energy depletion. Any method of sperm enrichment or isolationcan be used consonant with the invention, including density gradientcentrifugation, swim up, microfluidics, or a combination thereof

Sperm may be used in the methods provided by the invention either freshor from a preserved stock. For example, in some embodiments, prior totreatment, the sperm are recovered from cryogenic storage. In otherembodiments, prior to treatment, the sperm are recovered fromnon-cryogenic storage.

Different quantities of sperm can be used in the methods provided by theinvention, including fractions of a single ejaculate or a wholeejaculate. In some embodiments, the sperm are pooled from two or moreejaculates (e.g., 2, 3, 4, 5, 6, or more ejaculates).

Methods of Obtaining Sperm Sample

Various methods of collection of viable sperm are known. Such methodsinclude, for example, masturbation into sterile containers, thegloved-hand method, use of an artificial vagina, andelectro-ejaculation. Animal semen can be collected by using artificialvagina, electro-ejaculator, or by massaging the ampule of the animal byhand. It can also be directly collected from any section of the malereproductive tract including testicular sperm, and sperm obtained fromcaput, corpus or cauda epididymis using different methodologies such aspuncture of the testis or epididymis using surgical procedures orremoving the testis or epididymis and collecting the sperm insurrounding media. The sperm are preferably collected or quicklytransferred into an insulated container to avoid a rapid temperaturechange from physiological temperatures (typically about 35° C. to about39° C.). The ejaculate typically contains about 0.5 to 15 billion spermper milliliter, depending upon the species and particular animal. Thenumber may be reduced if obtained from a subfertile male or malesuffering from sperm dysfunction.

The sperm may be freshly collected sample from a source animal (e.g., amammal), or can be previously thawed or cryopreserved sample. At thetime of collection, or subsequently, the collected sperm may be combinedwith any of a number of various buffers that are compatible with sperm,such as TCA, HEPES, PBS, or any of the other buffers disclosed in U.S.Patent Application Publication No. US 2005/0003472, the content of whichis hereby incorporated herein by reference. For example, a bovine semensample typically containing about 0.5 to about 10 billion sperm cellsper milliliter may be collected directly from the source mammal into avessel containing a buffer to form a sperm suspension. The spermsuspension may also contain a range of other additives to maintain spermviability. Exemplary additives include protein sources, antibiotics,growth factors, and compositions that regulate oxidation/reductionreactions intracellularly and/or extracellularly. Examples of each ofthese additives are well known in the art, as demonstrated in thedisclosure of, for example, U.S. application Ser. Nos. 60/557,407 and11/092,313, the content of each of which is hereby incorporated hereinby reference. Alternatively, the semen sample may be collected into anempty container and then subsequently contacted with a buffer withinseveral minutes to hours after collection to form the sperm suspension.In some embodiments, the sperm cells can be collected directly into acontainer containing energy depletion medium (e.g., HTF medium devoid ofglucose, pyruvate and lactate) for incubation under energy depletion. Insome embodiments, the sperm cells can be collected in an empty containerand subsequently incubated under energy depleting conditions.

In some embodiments, sperm collection comprises washing sperm cellsprior to carrying out the methods disclosed herein. Generally, washinginvolves centrifuging a sample of semen or thawed sperm through adiluting wash media, which allows collection of a sperm-rich pellet.After a sperm wash process, or in place of it, a specific procedure forthe isolation of the motile sperm from a sample can be done.

In some embodiments, the sperms are isolated from semen prior to use inmethods disclosed herein. In some embodiments, sperm with increasedfunction can be further enriched, (for example, enriching sperm withincreased motility), from sperm prepared according to methods disclosedherein. Generally, sperm are isolated or enriched by allowing the motilesperm to swim away from the dead sperm, non motile sperm and debris(sperm swim-up), by centrifuging the sperm through a density gradient,or by passing the sperm through a column that binds the dead sperm anddebris. Isolating (or enriching) the spermatozoa from semen is performedby a method selected from the wash and spin method, the sedimentationmethod, the direct swim-up method, the pellet and swim-up method, andthe buoyant density gradient method. These methods are well known in theart. They are traditionally used in assisted reproduction techniques anddescribed in detail in “A textbook of In vitro Fertilization andAssisted Reproduction, The Bourn Hall guide to clinical and laboratorypractice, editor: Peter R. Brinsden, The Parthenon Publishing Group”(1999). In some embodiments, the sperm prepared by the methods disclosedherein can be further enriched for motile sperms by isolation proceduressuch as the sedimentation method, the direct swim-up method, the pelletand swim-up method, and the buoyant density gradient method.

The direct swim-up method implies self-selection of motile sperms,essentially comprising layering an aliquot of medium on top of a semensample or a preparation of sperm disclosed herein and allowing it tostand at room temperature for a certain period of time. The motile spermcells will migrate into the top layer (medium), from which they can berecovered. The method may also include centrifugation step(s). Theadvantage of “swim-up” selected spermatozoa is that the motile cellspresent in the sample are isolated and concentrated and that theproportion of morphologically normal sperm is increased.

The method may be varied and combined with further isolation/separationtechniques, depending on the amount of motile cells in the sample. Forexample, the swim-up procedure may be performed through the layering of1 ml of medium containing albumin on 1 ml of underlying seminal liquidin a test tube. After one hour of incubation at 37° C. in the air or in5% CO2 the upper phase of the medium to which the spermatozoa withbetter motility characteristics have migrated is collected. Thistechnique may also comprise or be combined with a centrifugation step,for example centrifugation on Percoll gradients. In typicalapplications, a sperm containing solution is layered over a gradientmaterial, preferably Percoll at 30-90% mixed with 0.05% pectin, and thensubjected to centrifugation to collect sperm enriched for improvedfunction. The separated, isolated or enriched spermatozoa are then usedin methods disclosed herein or may be cryopreserved before being furtherprocessed, for example. In case of the preparation of sperms prepared bymethods herein, they can be used for IVF, ICSI or artificialinsemination following enrichment steps or may be cryo-preserved forlater use, for example. Accordingly for any of these isolation, orenrichment methods, the sample may be semen, partially purified sperm,purified sperm, or sperm with increased function prepared by methodsherein. In some embodiments, the percentage of motile cells is increasedby at least 10%, at least 20%, at least 50%, at least 75%, at least 80%,or about 100% after isolating or enriching the sperm using isolationmethods, such as direct swim up, the pellet and swim-up method, and thebuoyant density gradient method compared to untreated semen sample orunenriched sperm preparation.

In some embodiments, after isolation, enrichment and washing, the spermpellet can be resuspended in a medium suitable for further processing,including preservation medium, HTF medium for culturing, medium forenergy depleting conditions (e.g., HTF devoid of glucose, lactate andpyruvate). As it relates to sperm with increased function prepared bymethods disclosed herein, the sperm preparation can be resuspended inpreservation medium, HTF medium for culturing, medium for insemination,assays of fertilization potential as described herein, in vitrofertilization, freezing, intrauterine insemination, cervical capinsemination, and the like. The sperm may be added to medium or themedium can be added to the sperm. The medium can be balanced saltsolution which may contain zwitterionic buffers, such as TES, HEPES,PIPES, or other buffers, such as sodium bicarbonate. In general, themedium for diluting sperm or culturing sperm, oocytes, embryos orembryonic stem cells is a balanced salt solution, such as M199,Synthetic Oviduct Fluid, PBS, BO, Test-yolk, Tyrode's, HBSS, Ham's F10,HTF, Menezo's B2, Menezo's B3, Ham's F12, DMEM, TALP, Earle's BufferedSalts, CZB, KSOM, BWW Medium, and emCare Media (PETS, Canton, Tex.). Insome embodiments, TALP or HTF is used for sperm culture medium, and CZBis used for embryo culture medium. The sperm, or embryo of the presentdisclosure can be preserved in a cryogenic medium comprising acryoprotectant.

Suitable Control Sperm

A suitable control sperm can be sperm incubated under controlconditions, i.e., in a control buffer such as, human tubal fluid (“HTF”)medium or modified HTF medium and not in energy depletion conditions.HTF comprises a sodium bicarbonate buffering system and may be utilizedfor uses requiring a carbon dioxide atmosphere during incubation.Modified HTF comprises a combined sodium bicarbonate and HEPES([4-2(2-hydroxyethyl)-1-piperazineethanesulfonic acid]) buffer. Suitableexamples of HTF medium or modified HTF medium include those that arecommercially available from Irvine Scientific, Santa Ana, California. Insome embodiments, the incubating in energy depletion conditions can beincubating the HTF medium from which glucose, lactate and pyruvate hasbeen omitted. The sperm may be incubated for a period sufficient toprovide a measurable change in the motility (or other characteristics)of the sperm; in specific embodiments of the method, incubation is from1 minute to 24 hours, 15 minutes to 3 hours, 30 minutes to 1.5 hours,about 1 hour, or any subrange or subvalue thereof. In some embodiments,a suitable control sperm is sperm which is incubated in energy depletionconditions followed by treatment with a first energy source (e.g.,selected from a gluconeogenesis substrate, or a glycolytic substrate) ora second energy source (e.g., selected from a gluconeogenesis substrate,or a glycolytic substrate but not same as first energy source)independently. In some embodiments, a suitable control sperm is spermwhich is incubated in energy depletion conditions followed by treatmentwith a gluconeogenesis substrate, or a glycolytic substrateindependently. In some embodiments, a suitable control is a sperm whichis incubated in energy depletion conditions followed by treatment with afirst energy source and a second energy source simultaneously. In someembodiments, a suitable control sperm is a sperm which is incubated inenergy depletion conditions followed by treatment with a gluconeogenesissubstrate and a glycolytic substrate simultaneously. In someembodiments, a suitable control sperm is an untreated sperm. It isunderstood that a suitable control sperm can be at least one sperm or apopulation of sperm, for example, a sperm preparation, or a spermsuspension.

Sperm Preparation

In some embodiments, the invention provides sperm preparations, such aspreparations of activated (e.g., sperm having been starved followingintroduction of an effective amount of both the first and second energysources, enriched for hyperactivated and intermediate sperm), partiallyactivated sperm (sperm having been starved and contacted with aneffective amount of only a first energy source), or potentiated sperm.These are collectively “sperm preparations provided by the invention” or“preparations provided by the invention.” In some embodiments theinvention provides preparations of hyperactivated sperm comprising atleast 5% hyperactivated sperm , e.g., at least about: 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0,13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0,19.5, 20.0%, or more hyperactivated sperm, e.g., between about: 5-20,8.5-20, 10-20, or 12.5-20%. In some embodiments, the preparation alsocontains at least about: 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, or 30% intermediatemotility sperm, e.g., between about 20.5-30%, 22.5-30%. Thus, in someembodiments, the percentage sum of hyperactivated and intermediatemotility sperm is at least: 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5,20.0, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25, 26, 27, 28,29, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50%, or more, e.g.,between about: 10-50, 30.5-50, 32.5-50. As the skilled artisan willappreciate, sperm may be separated based on hyperactivation (and/orintermediate) phenotype, but in some embodiments, the foregoingpercentages are based on preparations that have not been activated andthen sorted based on hyperactivation (however, in some embodiments,sperm preparations may have been pre-processed, e.g., to separate orotherwise enrich sperm from other seminal components, including certainirregular sperm). In some embodiments, the hyperactivated (orintermediate motility, or hyperactivated and intermediate motility)sperm in the preparation have 10, 15, 20, 25, 30, 35, 40, 45, 50%, ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, or more) reduction inintracellular RNA concentration (such as small non-coding RNAs,including microRNA), relative to a suitable control. In some embodimentssperm in a preparation provided by the invention are characterized (asassessed by either bulk average metrics or percentages in categories) byaltered sperm head morphology, increased tail movement (e.g.,amplitude), or a combination thereof

In some embodiments, the invention provides a preparation of spermprepared by any one of the methods provided by the invention.

In some embodiments, the invention provides preparations of spermprepared by enriching sperm from semen of a male subject, such as anormospermic male, sub fertile male, or oligospermic male, e.g., asubfertile (including oligospermic) male, incubating the sperm underenergy depletion for a time suitable to potentiate the sperm andproviding the sperm with a first energy source selected from: aneffective amount of a glycolytic energy source or an effective amount ofa gluconeogenesis substrate, but not an effective amount of both aglycolytic energy source and gluconeogenesis substrate.

For any of the preparations provided by the invention, sperm can be fromany male subject, such as a mammal, and in some embodiments, a human. Insome embodiments, the human is a normosperic male, or in otherembodiments, the male is an oligospermic or subfertile (e.g., low spermmotility) subject.

Promoting Fertilization

The preparation of sperm with increased function prepared by the methodsdisclosed herein can be useful to promote fertilization. Accordingly,the present disclosure relates to a method of promoting fertilization.The method comprises incubating a sperm under energy depletingconditions to potentiate the sperm, providing the potentiated sperm witha first energy source and a second energy source in a serial manner toincrease one or more sperm function, and providing the sperm withincreased function with access to an egg under conditions to promotefertilization. The preparation of sperm with increased function can beapplied in IVF, ICSI, artificial insemination (e.g., intra-uterineinsemination) in human as well as in the biomedical research industry ofanimal models for human diseases (infertility, sperm dysfunction), andin the breeding and agricultural industries. The sperm with increasedfunction prepared by the methods disclosed herein, can be providedaccess to an unfertilized egg of the same species as the sperm topromote in vitro fertilization, ICSI, or can be used for artificialinsemination, including for example, intrauterine insemination of femalesubjects of the same species as the sperm.

In Vivo Fertilization

The sperm with increased function prepared by the methods discloseherein can be useful to promote fertilization in vivo by providing thesperm with increased function access to an egg in the reproductive tractof a female subject of the same species as the sperm. In vivofertilization can be done by artificial insemination of sperm, forexample, by intracervical insemination or intrauterine insemination.Standard artificial insemination and intrauterine insemination, andother methods are well known to those of skill in the art. In someembodiments, the sperm with increased function is provided access to anegg in the reproductive tract of a female subject by intrauterineinsemination of the said sperm to promote fertilization of the egg. Inother embodiments, the sperm can be provided the second energy sourceand access to an egg in vivo by intrauterine insemination of a mammaliansperm which has been incubated under energy depletion conditions andprovided the first energy source in vitro. The sperm that is injected,may be used as held in suitable liquids. Liquid used for this purposemay be those liquids generally used as a medium for artificialinsemination.

In Vitro Fertilization

The present methods and preparation of sperm disclosed herein is ofparticular benefit in promoting fertilization by assisted reproductivetechnology, e.g., embryo viability following ART, and in particular IVF.Other suitable ART techniques to which the present disclosure isapplicable include, but are not limited to, gamete intrafallopiantransfer (GIFT), zygote intrafallopian transfer (ZIFT), blastocysttransfer (BT), intracytoplasmic sperm injection (ICSI), gamete, embryoand cell cryopreservation, in vitro preparation of embryos for embryobiopsy and other forms of embryo micromanipulation including formationof embryos by nuclear transfer and production transgenic lines andgenetically modified lines. It is also applicable to production ofembryonic stem cell lines.

In some embodiments, the sperm with increased function prepared by themethods disclosed herein can be used to fertilize an egg in vitro, suchas for example, by microinjection, including intracytoplasmic sperminjection (ICSI), and other methods well known to those in the art.Typically, in IVF, after fertilization, the cells are grown to theblastocyst stage and then implanted. The methods disclosed herein resultin increase in formation of an embryo with longer viability andincreased ability to develop into a 2-cell stage, blastocyst stage.Accordingly, the preparation of sperm disclosed herein can be useful invitro fertilization procedures, including, for example ICSI.

The methods of the present disclosure encompass providing the spermprepared by methods herein with access to an egg to promote in vitrofertilization. Providing the sperm access in vitro to the egg may becarried out in an appropriate medium. The medium used for this purposecan be a medium generally used as a medium for in vitro fertilization,for example, HTF medium. Temperature conditions for providing access maybe a general temperature to be used in vitro fertilization, for example,can be an average body or a temperature close thereto of the mammal.Time for providing access may be any time that is generally required invitro fertilization, but not particularly limited, and preferably from 6to 24 hours. In vitro fertilization rate can be determined by incubatingone or more sperms with matured oocytes for about 24 hr. Oocytes arethen be stained with a 1% aceto-orcein stain to determine the percentfertilized, or left in culture to divide and the number of embryosformed are counted. Oocytes can be matured in vitro in M199 media with50 μg luteinizing hormone/ml (Brackett and Zuelke, Theriogenology 39:43,1993)

Fertilization Uses

These methods and preparation of sperm disclosed herein are generallyapplicable to many species, including human, bovine, canine, equine,porcine, ovine, avian, rodent and others. Although useful wheneverfertilization is desired, the present methods have particular use inanimals and humans that have a fertilization dysfunction in order toincrease the likelihood of conception. Such dysfunctions include lowsperm count, reduced motility of sperm, and abnormal morphology ofsperm. Accordingly, the methods disclosed herein can be useful forpreparation of sperm with increased function in infertility clinicsprior to their use in vitro fertilization or intrauterine insemination.The methods described herein can be used to improve artificialinsemination, IVF or ICSI in exotic species and/or endangered species.As such the methods can find use for promoting fertilization in animalsmaintained captive in a zoo, and in conservation programs aiming toimprove reproduction in animals that are close to extinction in thewild. For example, the methods and preparation of sperm of the presentdisclosure can be used to improve fertilization and pregnancy rates inanimal husbandry, for species of agricultural value, and in species bredfor conservation purposes.

In addition, the methods and compositions of the present invention areuseful in artificial insemination procedures, e.g., in commercialbreedings. The method can be carried out with sperm from domesticatedanimals, especially livestock, as well as with sperm from wild animals(e.g., endangered species). For example, as disclosed herein,embodiments of the methods and compositions of the disclosure findapplication in bovine reproduction. The methods and preparation can beuseful for artificial insemination in the livestock production industrywhere it is desirable to influence the outcome towards offspring havingone or more preferred characteristics or traits by introducing specificgenetically-determined traits into the livestock, e.g., offspring of aparticular gender, offspring with enhanced milk production, offspringfor quality meat production. Use of the methods described herein willresult in improved pregnancy rates. Mammalian sperm are frequentlydamaged by freezing and thawing and results in lower fertility. Byimproving the performance of the viable sperm, sperm prepared by methodsdisclosed herein when used for insemination may promote a higherpregnancy rate per estrus cycle, reducing the number of cycles requiredto ensure conception and hence reducing the overall cost of artificialinsemination.

Semen from animals with highly desirable traits could be used toinseminate more females because fewer cycles would be needed to ensureconception in any one female. For such applications, the semen isobtained from a male with desired characteristics. In order to influencegender outcome of the resulting offspring, the sperm preparation can besorted into X- and Y chromosome bearing cells, and/or enriched for spermwith one or more increased sperm function disclosed herein. The spermmay be sorted by commonly used methods, for example, as described inJohnson et al. (U.S. Pat. No. 5,135,759) using a flow cytometer/cellsorter into X and Y chromosome-bearing sperm enriched populations. Thesperm prepared by the methods disclosed herein can be sorted the into apopulation comprising a certain percent X chromosome bearing or Ychromosome bearing sperm cells. For example, the spermatozoa of one ofthe populations may comprise at least about 65% X chromosome bearing orY chromosome bearing sperm cells, at least about 70% X chromosomebearing or Y chromosome bearing sperm cells, at least about 75% Xchromosome bearing or Y chromosome bearing sperm cells, at least about80% X chromosome bearing or Y chromosome bearing sperm cells, at leastabout 85% X chromosome bearing or Y chromosome bearing sperm cells, atleast about 90% X chromosome bearing or Y chromosome bearing spermcells, or even at least about 95% X chromosome bearing or Y chromosomebearing sperm cells. In some embodiments, the sorting can be done priorto preparing the sperm with increased function as disclosed herein. Insome embodiments, the sorting can be done prior to providing the spermwith increased function with access to an egg for fertilization as inIVF, ICSI or AI.

The methods and preparations provided by the invention can be used inassisted fertilization, such as IVF, including by ICSI (intracytoplasmicsperm injection). In some embodiments, any of the methods provided bythe invention can include the step of providing the sperm to a femalereproductive tract, optionally wherein the effective amount of a secondenergy source is provided in the female reproductive tract. In someembodiments, a sperm preparation provided by the invention (havingincreased sperm function) can be provided access to an egg for a timesufficient to fertilize the egg, which egg may be ex vivo (e.g., IVF,including ICSI) or, in some embodiments, in a female reproductive tract.Such methods, in some embodiments, entail a subsequent implantation ofthe fertilized egg in a female carrier.

In some embodiments, the invention provides methods of fertilizationcomprising providing a preparation provided by the invention that hasnot been contacted with an effective amount of a second energy sourcewith access to an egg and an effective amount of a second energy sourceso as to provide an effective amount of both a gluconeogenesis substrateand a glycolytic energy source for a time sufficient to fertilize theegg. In some embodiments, these methods are performed in vitro. In otherembodiments, these methods are performed in vivo, in the reproductivetract (vagina or uterus) of a female.

Articles of Manufacture

In some embodiments, the invention also provides articles of manufactureand kits, e.g., suitable for performing any of the methods provided bythe invention or preparing any of the preparations provided by theinvention. For example, in some embodiments, the invention providesarticles of manufacture comprising a sperm potentiating solution that,upon contact with sperm, induces energy depletion; a first solutionproviding a first energy source selected from: an effective amount of aglycolytic energy source or an effective amount of a gluconeogenesissubstrate, but not an effective amount of both a glycolytic energysource and gluconeogenesis substrate; and a second solution providing aneffective amount of a second energy source. In some embodiments, thearticles of manufacture further include a sperm isolating matrix. Inmore some embodiments, the sperm isolating matrix is silanized silica,optionally wherein the silanized silica is in media substantially freeof any glycolytic energy source or gluconeogenesis substrate. In someembodiments, the kit comprises instructions for carrying out the methodsdisclosed herein. The kit can also include a washing medium, apreservation medium, culture medium (e.g., HTF), a diluent, and thelike. The kits can further contain adjuvants, reagents, and buffersnecessary.

The kits can also include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements, such as the potentiating solution, first solutionproviding the first energy source, and second solution providing thesecond energy source to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers can be formed from a variety of materials such asglass or plastic. The articles of manufacture provided herein containpackaging materials. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes, bags,containers, bottles, and any packaging material suitable for use inmethods disclosed herein. A kit typically includes labels listingcontents and/or instructions for use, and package inserts withinstructions for use. A set of instructions can also be included.

EXAMPLES

The present disclosure will be described in greater detail by way of thefollowing specific examples. The following examples are offered forillustrative purposes, and are not intended to limit the invention inany manner. Those of skill in the art will readily recognize a varietyof non-critical parameters that can be changed or modified to yieldalternative embodiments according to the invention. All patents, patentapplications, and printed publications listed herein are incorporatedherein by reference in their entirety.

Example 1: Materials and Methods Media

Media for human sperm capacitation was Human Tubal Fluid (Complete HTFor C-HTF) medium, containing 97.8 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.37mM KH2PO4, 0.2 mM MgSO4. 7H2O, 25.1 mM NaHCO3⁻, 0.33 mM Na-pyruvate,2.78 mM glucose, lactate 21.4 mM and 5 mg/mL human serum albumin (HSA),10 μg/mL gentamicin and phenol red 0.0006% at pH 7.4 equilibrated with5% CO2. For sperm starvation treatment glucose, lactate and pyruvatewere omitted from the HTF media above (F-HTF, test media).

Semen Samples

Semen samples were obtained from healthy males or males seekingtreatment for infertility by masturbation into sterile containers.Ejaculates were liquified for up to 2 hours prior to processing for theexperiment. Following liquefaction, the volume of the ejaculate wasdivided equally for processing into F-HTF (test) conditions or C-HTF(control conditions). Semen samples were processed by eitherdensity-gradient centrifugation or direct swim up method to collectviable sperm cells.

Sperm Processing Density Gradient Centrifugation

Following liquefaction, the entire volume of each ejaculate was equallydivided over two different gradient conditions. The test sample wasprepared using a 45-90% Percoll (Sigma, P-1644)) gradient in phosphatebuffered saline. The control sample was prepared using an Isolategradient (Irvine Scientific, Santa Ana, Calif.; 99264) in human tubalfluid. Both samples were centrifuged for 20 min at 500×g. Followingcentrifugation, the supernatant was removed, and the pellet washed with10 ml media. The test sample was washed in F-HTF and the control samplewas washed in C-HTF.

Sperm Swim Up

Following liquefaction, the entire volume of each ejaculate was dividedinto a test sample and control sample, as previously described. The testsample was layered gently with 2.5 ml of F-HTF. The control sample waslayered with C-HTF medium. Tubes were carefully inclined at a 45° angleand incubated for 1 h at 37° C., 5% CO2. The supernatant was carefullycollected, and washed F-HTF and the control sample was washed in C-HTF.

Analysis of Sperm Motility

Sperm suspensions of test and control sperm (6 μl) were loaded into onepre-warmed chamber slide (depth, 20 μm) (Leja slide, SpectrumTechnologies) and placed on a microscope stage at 37° C. Sperm motilitywas examined using the CEROS II computer-assisted semen analysis (CASA)system (Hamilton Thorne Research, Beverly, Mass.). One-second trackswere captured using the following settings: 60 frames per second, 60frames acquired, minimum contrast=80, minimum size=3 pixels, defaultcell size=6 pixels, default cell intensity=160, slow cells counted asmotile, low VAP cutoff=10 μm/s, low VSL cutoff=0 pm/s, minimum intensitygate=0.18, maximum intensity gate=1.21, minimum size gate=0.56 pixels,maximum size gate=2.63 pixels, minimum elongation gate=0 pixels, andmaximum elongation gate=99 pixels. Raw data were sorted and analyzedusing the CASAnova parameters (Goodson et al., 2018, supra). At least 20microscopy fields corresponding to a minimum of 500 sperm were analyzedin each experiment.

Example 2: Experimental Results

This example shows that serial reintroduction of energy source afternutrient depletion increases sperm hyperactivation.

Incubating sperm in a glucose, pyruvate and lactate-free media for threehours resulted in a reduction in motility as shown in FIG. 1. Rescue ofsperm motility was tested with different energy substrates. When spermwere starved for 3 hour and rescued with a complete HTF spermhyperactivation and intermediate motility were elevated compared withthe control treatment (FIG. 2). In contrast, sperm treated with glucose(5 mM) or pyruvate (0.33 mM) alone did not improve sperm hyperactivationcompared to the control (FIG. 2). Reintroduction of pyruvate alone hadno impact on sperm motility from the starvation state, however,reintroduction of glucose alone restored motility to the levels ofcontrol (FIG. 2) suggesting that glucose is the major energy sourcerequired for sperm hyperactivation. Surprisingly, when pyruvate wasadded to the glucose-treated sperm or glucose to the pyruvate-treatedsperm, this triggered a significant elevation in hyperactivationmotility relative to control conditions or when both pyruvate andglucose were reintroduced to sperm at the same time

Example 3: Enhancing Activation

Osmolarity of C-HTF is approximately 290 mOsm, where F-HTF isapproximately 243 mOsm. To illustrate that hypotonic conditions stresssperm such that when reversed, triggers elevated sperm motility andfunction, sperm are incubated in different conditions that arehypotonic, isotonic, or hypertonic in the presence of a carbon sourcethat is not metabolized efficiently by the sperm such as trehalose,dextran, or other long chain sugar, and impacts on motility observed byCASA analysis during incubation in hypotonic conditions and followingreturn to isotonic conditions. This includes adjusting concentrations ofvarious ions such as calcium, sodium, and potassium during thepotentiation phase, and evaluating motility following return to C-HTF.Additionally, impacts of increasing or decreasing the concentration ofions such as calcium, sodium and potassium during both the potentiationphase and the rescue phase are tested, as are the staged addition ionsto mimic the ion cycling that occurs in the female reproductive tractduring natural conception. In addition to motility, calcium ion flux isassessed. These manipulations, either alone or in conjunction with thedescribed manipulation of glucose and pyruvate enhance the percentage ofsperm that achieve hyperactive or intermediate motility.

Although human sperm exhibit reduced motility during the starvationphase of these treatments, the sperm do not completely stop movingsuggesting that the cells are utilizing an internal energy source suchas glycogen or degrading cellular components such as lipids, proteins,or RNA. Sperm exposed to the starvation phase are assessed for totallipid content, RNA content, and protein content. Proteomic, metabolomic,and lipidomic analysis are performed following the starvation phase,following addition of first energy source, and following addition ofsecond energy source to illustrate intracellular changes associated withsperm motility states. Total RNA (including certain subfractions, suchas mRNA or small non-coding RNA, such as microRNA) is measured in spermtreated with control conditions and sperm treated with test conditions,as illustrated in Example 2. The results of this analysis will indicateRNA is being used as an energy source by sperm.

Staging introduction of upstream carbon sources for glycolysis (such asglucose, mannose, fructose, dextrose, or sucrose) and downstreammetabolites (such as pyruvate, lactate, succinate, citrate, fumarate,malate) change the rate of conversion of AMP to ATP resulting inimproved sperm motility and function as compared to simultaneousaddition. ATP and AMP levels are measured in sperm following starvation,introduction of first energy source and introduction of second energysource. Staged introduction of nutrients following starvation increasesconversion of AMP to ATP.

Example 4

This example provides additional evidence that staged reintroduction ofenergy sources activates sperm.

Sperm samples from men seeking treatment for infertility were obtainedfrom a fertility clinic. These samples included normally fertile andsubfertile sperm. To improve sperm quality, samples were prepared bydensity gradient centrifugation as described in Example 1. Followingliquefaction, the entire volume of each ejaculate was equally dividedand subjected to two different density gradient conditions. The testsample was prepared using a 45-90% Percoll (Sigma, P-1644) gradientdiluted in phosphate buffered saline solution devoid of nutrients with afinal pH of 7.4 (F-PERCOLL). The control sample was prepared using a45-90% Percoll gradient diluted in phosphate buffered saline solutionwith nutrients such as (lactate, glucose and pyruvate) with a final pHof 7.4 (C-PERCOLL). Both samples were centrifuged for 20 min at 500×g.Following centrifugation, the supernatant was removed, and the pelletwashed with 10 ml media. The test sample was washed in F-HTF and thecontrol sample was washed in C-HTF.

Samples were treated with C-HTF media as described in example 1 orseparated by density gradient in a nutrient free media and washed withF-HTF. Sperm with F-HTF A) 1 hour incubation in F-HTF followed byaddition of glucose (5 mM), pyruvate (0.33 mM) and lactate incubationfor 1 hour 15 minutes, B) 1 hour incubation in F-HTF, addition ofpyruvate for 1 hour, then addition of glucose for 15 minutes, or C) 1hour incubation in F-HTF, addition of glucose for 1 hour then additionof pyruvate for 15 minutes. Samples were analyzed by CASA as outlined inExample 1. Results are shown in FIGS. 5A and 5B, and FIGS. 6A and 6B.Each test condition resulted in an increase in the number of sperm withintermediate and hyperactive motility relative to control, with thehighest level of activation observed with treatments B and C.

To speed up the starvation state, sperm were separated by densitygradient in a nutrient free media and washed with 10 ml F-HTF. After1-hour incubation in F-HTF, sperm with reduced motility similar to thereduced motility as seen in FIG. 1 were primed with either pyruvate(0.33 mM) or glucose (5 mM) for one hour and then rescued with either(B) glucose (5 mM) or (C) pyruvate (0.33 mM) for 15 minutes as depictedin FIG. 3. Similar to the results shown in FIG. 2, this speed/starveprotocol also significantly improved the sperm motility parameters shownin FIG. 5

Example 5

This example describes use of sperm treated according to certainembodiments of the invention to improve fertility in human subjectsundergoing IUI.

Subjects are adult females (e.g., between 18 and 35 years old) withouthistory of recurrent pregnancy loss and may or may not having previouslyattempted IUI. Subjects are treated with standard of care medicines(e.g., Clomid preparation, with Hcg triggering injection as indicated)and randomly assigned to receive either IUI of sperm prepared bydiluting and centrifuging semen on C-HTF or F-HTF and collecting andresuspending cells in C-HTF or F-HTF. Alternatively, the sperm can becollected by density gradient centrifugation and washing andresuspending cells in C-HTF or F-HTF. Sperm are treated with F-HTF(e.g., for 1 hour), then either pyruvate or glucose is added and thesperm are incubated (e.g., for 1 hour), and then the sperm are used forinseminating the female. Sperm are treated with C-HTF (e.g., 2 hours),and then the sperm are used for inseminating the female. Pregnancies aremonitored with regular follow-up. Females receiving sperm incubated inthe absence of glucose (e.g., 5mM) or pyruvate (e.g., 0.33 mM) followedby the staged addition of glucose or pyruvate are expected to exhibit aparameter of improved fertility, for example, increased rate ofpregnancy, fetal heart rate (e.g., at 7 weeks), ongoing pregnancy (e.g.,at 10 weeks) and/or livebirth rates.

Example 6

This example describes use of sperm treated according to certainembodiments of the invention to improve fertility in human subjectsundergoing IVF.

Subjects are adult female subjects (e.g., between 18 and 35 years old)without history of recurrent pregnancy loss and may or may not havingpreviously attempted IVF. Subjects are treated with standard of caremedicines (e.g., ovulation suppression followed by ovulationstimulation, with Hcg triggering injection as indicated) prior to eggretrieval. Subjects are randomly assigned to the control group or thetreatment group. In the control group, sperm are collected by densitygradient centrifugation are resuspended in either sperm wash media,C-HTF or Fertilization media. Non-limiting examples of commerciallyavailable fertilization media include Global Total for fertilization(Origio), Continuous Single Culture®-NX Complete (Irvine), Sydney IVFFertilization Medium (Cook Medical). In the treatment group, sperm arecollected by density gradient centrifugation, are washed and resuspendedin F-HTF for sufficient incubation to potentiate the sperm (e.g., 1hour). Following this incubation, either pyruvate (0.33 mM) or glucose(5 mM) is added and the sperm are incubated (e.g., for 1 hour).Following this incubation, either glucose (5 mM) or pyruvate (0.33 mM)(whichever was not added in the first step) is added and the sperm areincubated (e.g., at least 15 minutes). For both the treatment andcontrol groups, sperm will be incubated with eggs in vitro andfertilization rates and embryo development monitored. Embryos (e.g., atDay 5) will be transferred to the female and pregnancy will bedetermined by blood test (e.g., 2 weeks later). Pregnancies aremonitored with regular follow-up. Females receiving embryos generatedwith sperm incubated in the absence of glucose and pyruvate followed bythe staged addition of glucose and pyruvate are expected to exhibit animproved parameter of fertility, e.g., increased rates of fertilization,blastocyst development, pregnancy, fetal heart rate (e.g., at 7 weeks),ongoing pregnancy (e.g., at 10 weeks) and/or livebirth rates.

It should be understood that for all numerical bounds describing someparameter in this application, such as “about,” “at least,” “less than,”and “more than,” the description also necessarily encompasses any rangebounded by the recited values. Accordingly, for example, the description“at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2,1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, or other reference cited herein, such asnon-patent literature and reference sequence information, it should beunderstood that they are incorporated by reference in their entirety forall purposes as well as for the proposition that is recited. Where anyconflict exists between a document incorporated by reference and thepresent application, this application will control. All informationassociated with reference gene sequences disclosed in this application,such as GeneIDs or accession numbers (typically referencing NCBIaccession numbers), including, for example, genomic loci, genomicsequences, functional annotations, allelic variants, and reference mRNA(including, e.g., exon boundaries or response elements) and proteinsequences (such as conserved domain structures), as well as chemicalreferences (e.g., PubChem compound, PubChem substance, or PubChemBioassay entries, including the annotations therein, such as structuresand assays, et cetera), are hereby incorporated by reference in theirentirety.

Headings used in this application are for convenience only and do notaffect the interpretation of this application.

Preferred features of each of the aspects provided by the invention(e.g., media, compositions, preparations, and methods) are applicable toall of the other aspects of the invention mutatis mutandis and, withoutlimitation, are exemplified by the dependent claims and also encompasscombinations and permutations of individual features (e.g., elements,including numerical ranges and exemplary embodiments) of particularembodiments and aspects of the invention, including the workingexamples. For example, particular experimental parameters exemplified inthe working examples can be adapted for use in the claimed inventionpiecemeal without departing from the invention. For example, formaterials that are disclosed, while specific reference of each of thevarious individual and collective combinations and permutations of thesecompounds may not be explicitly disclosed, each is specificallycontemplated and described herein. Thus, if a class of elements A, B,and C are disclosed as well as a class of elements D, E, and F and anexample of a combination of elements A-D is disclosed, then, even ifeach is not individually recited, each is individually and collectivelycontemplated. Thus, in this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-groups of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this application, including elements of acomposition of matter and steps of method of making or using thecompositions.

The forgoing aspects of the invention, as recognized by the personhaving ordinary skill in the art following the teachings of thespecification, can be claimed in any combination or permutation to theextent that they are novel and non-obvious over the prior art—thus, tothe extent an element is described in one or more references known tothe person having ordinary skill in the art, they may be excluded fromthe claimed invention by, inter alia, a negative proviso or disclaimerof the feature or combination of features.

What is claimed is:
 1. A method for preparing a mammalian sperm for usein an assisted reproductive procedure, the method comprising; (a)incubating the mammalian sperm under energy depletion conditions for atime suitable to generate a potentiated mammalian sperm; and (b)providing the potentiated mammalian sperm from step (a) with aneffective amount of a first energy source and a second energy source ina serial manner, wherein the effective amount is the amount sufficientto induce improved sperm function, and wherein the mammalian spermprovided by step (b) is suitable for use in the assisted reproductiveprocedure.
 2. The method of claim 1, wherein one or more sperm functionis selected from curvilinear velocity, amplitude or lateral headdisplacement, autophagy, sperm capacitation, percentage ofhyperactivated sperm, percentage of intermediate motility sperm andpercentage of hyperactivated sperm and intermediate motility sperm, isimproved relative to a suitable control sperm.
 3. The method of claim 1,further comprising resuspending the mammalian sperm provided by step (b)in a fertilization medium, a preservation medium, or a culture medium.4. The method of claim 3, wherein the fertilization medium is humantubal fertilization medium.
 5. The method of claim 1, further comprisingthe step of cryopreserving the mammalian sperm provided by step (b)prior to use in the assisted reproductive procedure.
 6. The method ofclaim 1, wherein the assisted reproductive procedure comprises in vitrofertilization of an egg by the mammalian sperm provided by step (b) togenerate an embryo.
 7. The method of claim 1, wherein the assistedreproductive procedure is selected from the group consisting of frozenembryo transfer (FET), intracytoplasmic sperm injection (ICSI), gameteintrafallopian tube transfer (GIFT), and zygote intrafallopian tubetransfer (ZIFT).
 8. The method of claim 1, wherein the assistedreproductive procedure is artificial insemination of the mammalian spermprovided by step (b).
 9. The method of claim 8, wherein the artificialinsemination is intra-uterine insemination or intracervical inseminationof the mammalian sperm provided by step (b).
 10. The method of claim 1,wherein the mammalian sperm of step (a) is recovered from a cryogenicstorage.
 11. The method of claim 1, wherein the mammalian sperm of step(a) is recovered from a non-cryogenic storage.
 12. The method of claim1, wherein the mammalian sperm of step (a) is from an oligospermicsubject or a subfertile subject.
 13. The method of claim 1, wherein themammalian sperm of step (a) is a human sperm.
 14. The method of claim 1,wherein the mammalian sperm of step (a) is provided as a pool of two ormore ejaculates.
 15. The method of claim 1, wherein the mammalian spermof step (a) is enriched from semen prior to step (a) by density gradientcentrifugation, swim up, or microfluidics.
 16. The method of claim 1,wherein the first energy source is a glycolytic energy source and thesecond energy source is a gluconeogenesis substrate, or the first energysource is the glycolytic energy source and the second energy source isthe gluconeogenesis substrate.
 17. The method of claim 16, wherein theglycolytic energy source is glucose.
 18. The method of claim 16, whereinthe gluconeogenesis substrate is pyruvate.
 19. The method of claim 1,wherein the method is performed at an osmolality ranging from 200-280mOsm/kg.
 20. The method of claim 1, wherein the incubating under energydepletion conditions of step (a) is for at least 10 min.